Light sheet microscopy, or light sheet fluorescence microscopy (LSFM), is newly of interest to researchers, and for good reason. LSFM is a type of fluorescence microscopy in which the illumination and detection pathways are perpendicular to each other, which is key for understanding its benefits. The user illuminates the specimen with a thin sheet of light, which excites fluorophores in the optical section delineated by the light sheet. Fluorescence then is detected along a pathway perpendicular to the light sheet.
Some researchers are choosing LSFM over other imaging techniques such as wide-field and confocal microscopy, in which the sample is exposed to illumination light above and below the plane of focus. In LSFM, the out-of-focus fluorescence is virtually eliminated. The result is that compared to other methods, LSFM has dramatically lower amounts of phototoxicity and photobleaching, so that live tissue remains healthy for longer. LSFM can also optically section the specimen at a high speed for 3D imaging.
LSFM was already well-suited for imaging relatively transparent specimens, but recent advances in tissue clearing methods are widening its range of possible specimens. Increasingly, researchers are taking advantage of LSFM to reveal anatomic information of larger cleared tissues that were previously inaccessible. This article discusses examples of today’s LSFM systems and their applications.
Commercially available tools
Manufacturers of imaging equipment are starting to offer tools for researchers interested in LSFM. Leica Microsystems’ DLS module combines with a confocal microscope to shed light on specimens from different angles using different modalities. For light sheet imaging with DLS, Leica offers different detection objectives for the desired magnification and resolution, whether imaging subcellular structures with high resolution, or larger tissues at lower resolution. Additionally, “the confocal point scanner could be leveraged to photoconvert or photomanipulate the specimen to investigate the consequences of such system perturbations,” adds Petra Haas, product manager for confocal applications at Leica Microsystems.
The versatility of the DLS contributes to its wide range of applications. “Complex 3D cell cultures are leveraged to investigate cell behaviors upon exposure to different compounds under physiological conditions [using the DLS],” says Haas. Another example is studies of specific processes within living organisms, “such as inside a beating heart of a zebrafish embryo, or during neuronal communications based on Ca2+ activity,” she adds.
An important consideration for anyone performing LSFM is sample handling. Because the DLS uses an inverted microscope, it uses fairly conventional mounting techniques, and can image several specimens simultaneously. “Additional tools like U-shaped glass capillaries or chemically inert sample holders for various mounting media make multi-position imaging and tile scan set-up readily available,” says Haas.
Sample handling is also a feature addressed by Zeiss’s new Lightsheet 7, a fully contained boxed system for LSFM designed for easy use in imaging a wide range of sample types. “Sample mounting was, and sometimes still is, a bit of an art form,” says Courtney Akitake, product marketing manager for Lightsheet at ZEISS Research Microscopy Solutions. Often researchers got creative in positioning samples just right, so that they were oriented properly for the separate illumination and detection pathways. “Now customized hardware and easy-to-use software loading routines quickly get samples ready for imaging,” says Akitake.
The Lightsheet 7 images diverse samples including whole C. elegans and Drosophila, organoids, marine organisms, brains and other organs; studying whole organism development and morphogenesis in zebrafish, 3D cell cultures, and plants is common. “Another key adaptation of light sheet microscopes is the development of specialized optics to image optically cleared specimens in addition to live developmental organisms,” she says. Many Lightsheet 7 users are imaging large, optically cleared, whole organ explants such as mouse brains. “Using low magnification objectives, Lightsheet users can first get cellular resolution in an overview of large cleared tissues,” says Akitake. “They can then switch to high magnification objectives for subcellular resolution in a specific region of interest.”
Building a microscope around the sample
The lab of Jan Huisken, Director of Medical Engineering and Lead Investigator of Multiscale Imaging at the Morgridge Institute for Research, takes a unique approach to accommodating different sample geometries: they build LSFM microscopes around the sample. Huisken’s lab uses various LSFM techniques to study the development and morphogenesis of the zebrafish heart and other organs.
Their new LSFM platform, Flamingo, is a powerful microscope that is portable and modular. “This system will allow us to work with many collaborators around the world,” says Huisken. “The idea is to develop and assemble a bespoke, turn-key system for collaborators and send them the complete microscope.” These custom systems are built around the sample so that the resulting instrument is optimized for the project, whether studying single cells, whole embryos, or cleared mammalian organs. Huisken says that the custom-designed LSFM platforms provide “low phytotoxicity and the speed needed to image fragile and quickly developing biological specimens.”
Huisken hopes that their efforts with the Flamingo platform will help to further open science. “By developing a flexible, modular platform we are in a position to provide a bespoke system for any collaborator,” he says. “In particular, scientists who cannot afford a commercial system can get access to a cutting-edge instrument easily.”
Fighting disease with LSFM
The lab of Jonathan Liu, director of the Molecular Biophotonics Laboratory at the University of Washington, harnesses the power of LSFM to combat disease, imaging optically cleared clinical specimens such as biopsies and surgical excisions. “We are working to show that nondestructive 3D pathology, with LSFM, can lead to improved diagnosis and grading of disease aggressiveness, which will ensure that individuals receive the most optimal treatments,” says Liu. “These large clinical datasets are being analyzed with machine-learning tools to advance the field of computational 3D pathology.” The lab also works with other researchers to image nonclinical specimens, including engineered tissues, mouse brains, and plants.
The Liu lab developed “open-top” light sheet (OTLS) microscopes to accommodate large and multiple specimens with arbitrary geometries. “OTLS microscopes are versatile and easy to use, allowing one or more samples to be mounted much like a document is placed on a flat-bed scanner,” says Liu. They are working on a new design for multi-resolution imaging over a broad range of scales, similar to what 2X–50X microscope objectives can achieve in standard pathology microscopes. “This system will also enable deeper imaging than our previous systems, and will be less sensitive to refractive index mismatch, which means that a greater variety and combination of clearing protocols and sample-holder substrates can be used,” he says.
Liu sees progress in many areas of the LSFM field today. One advance is the ability to perform LSFM with a single primary microscope objective, also called oblique plane microscopy, for rapid volumetric imaging of living organisms. “Techniques have also been developed to achieve isotropic or near-isotropic resolution with LSFM, including axially swept illumination and fusion deconvolution techniques,” he says. LSFM is also benefiting from recent advances in machine learning, including deep learning; the result is reduced image noise, better spatial resolution, and the ability to segment and classify tissue structures.
Such ongoing advances in LSFM will only widen its applications. For example, Liu uses OTLS microscopy to image large preclinical and clinical specimens in 3D at high resolution within certain time frames, and doesn’t believe he could do this successfully without the LSFM technology. “Other 3D microscopy systems would struggle to provide the working distance—in this case, the imaging depth—as well as the convenient sample mounting and speed that are required for our clinical applications,” he says. As more researchers creatively adapt LSFM, tailoring it to their own research ends, further innovations are likely to emerge.